veterinary hematology || hematopoiesis

OVERVIEWSites of Blood Cell ProductionIn mammals, primitive hematopoiesis begins outside the body of the embryo in the yolk sac and shortly thereafter within the aorta-gonad-mesonephros (AGM) region of the embryo.42,130,139 Small clusters of hematopoietic stem cells (HSCs) have been identified attached to the endothelium of the yolk sac and the dorsal aorta. These HSCs and the associ-ated endothelial cells are produced by common embryonic stem cells known as hemangioblasts.34,74,150 In addition to HSCs, committed erythroid and megakaryocytic progenitor cells, primitive erythrocytes (large nucleated cells containing embryonal hemoglobin), large primitive reticulated platelets, and rare primitive macrophages are also produced in the yolk sacs of rodents and humans.98,136 Notably, these primitive macrophages appear to develop directly from progenitor cells in the yolk sac without passing through a monocyte stage.14 Definitive erythropoiesis, prominent megakaryocytopoiesis, and limited leukocyte production also occur in the yolk sacs of cats, with hematopoiesis persisting longer during gestation than it does in rodents and humans.134 The AGM region transiently supports the development of HSCs and some committed hematopoietic progenitor cells (HPCs), but rec-ognizable blood cells are not produced in the AGM.93

Sites of blood cell production shift during embryonic and fetal development as optimal microenvironments are produced in various tissues (Fig. 3-1).102 The liver and, to a lesser extent, the spleen become the major hematopoietic organs by midges-tation in the fetus.129,134 Current evidence suggests that the AGM is more important than the yolk sack in providing HSCs to seed the liver and spleen, but the relative importance of each area in embryonic and fetal hematopoiesis remains to be clari-fied.102 Blood cell production begins in bone marrow and lym-phoid organs during midgestation in mammals, with nearly all blood cells being produced in these organs at the time of birth.134 Blood cells are produced in the bone marrow of adult birds20; the bone marrow and sometimes the spleen of adult reptiles30; the kidney, liver, spleen, and/or bone marrow of amphibians5,42; and the kidney and/or spleen of fish.42,48

Organization of Bone MarrowBone marrow develops in mammals during the second trimes-ter.21 Rudimentary fetal bone is initially filled with cartilage. Chondrocytes hypertrophy and promote mineralization of the cartilage matrix in the center of the rudimentary bone. This is followed by the entry of progenitor cells, which develop into chondroclasts that partially degrade the mineralized cartilage and form bone marrow spaces colonized by incoming blood vessels.23,131 Osteoblast progenitors enter the space created, adhere to remaining cartilage, develop into mature osteoblasts, and begin the formation of bony trabeculae. Vascular sinuses and extravascular mesenchymal cells subsequently form a con-nective tissue meshwork within which HSCs originating from the liver (and probably the spleen) bind, proliferate, and dif-ferentiate, ultimately producing circulating blood cells.131 When these structures are fully developed, blood is supplied to the bone marrow by nutrient arteries and periosteal capil-laries (Fig. 3-2).7

The stroma of the marrow is a connective tissue consisting of stromal cells (fibroblast-like cells, also called reticular cells), adipocytes, vascular elements (endothelial cells and myocytes), neural elements, and extracellular matrix (ECM), with the arrangement creating both intravascular and extravascular spaces (Figs. 3-3, 3-4).146,147 In postnatal mammals, blood cells are continuously produced within the extravascular spaces of bone marrow. Leukocytes are also produced within the extra-vascular spaces of bone marrow in birds, but erythrocytes and thrombocytes are produced within the vascular spaces of the avian marrow.20

This specialized arrangement of the marrow vasculature is important in the organization of intramedullary hematopoi-etic microenvironments, as marrow endothelial cells are actively involved in the regulation of transendothelial (not interendothelial) movement of hematopoietic cells and blood cells between the extravascular hematopoietic space and peripheral blood.92,114 Together, endothelial cells and stromal cells produce the ECM, which consists of collagen fibers, various macromolecules capable of binding cells, and basal laminae of the sinuses.100,109 The marrow stromal cells have

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FIGURE 3-1 Sites of definitive hematopoiesis during prenatal development in cats. Percentages represent relative contributions of sites to definitive blood cell production. Lymphoid populations also develop in lymph nodes and thymus beginning in midgestation (not shown).

Redrawn from Tiedemann K, van Ooyen B. Prenatal hematopoiesis and blood characteristics of the cat. Anat Embryol (Berl). 1978;153:243-267.

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FIGURE 3-2 Anatomy and circulation of the bone marrow. Periosteum (p), cortical bone (cb), nutrient foramen (nf ), nutrient artery (na), nutrient vein (nv), central longitudinal artery (cla), central longitudinal vein (clv), periosteal capillaries (pc), arteriole (a), sinuses (s), hematopoietic compartment (h), anastomosis of the nutrient capillaries and sinuses (1), anastomosis of the nutrient artery capillaries and periosteal capillaries (2), anastomosis of the periosteal capillaries and sinuses (3).

From Alsaker RD. The formation, emergence, and maturation of the reticulo-cyte: a review. Vet Clin Pathol. 1977;6(3):7-12.

extensively branched cytoplasmic processes and, along with the fibers that they produce, provide structural support for the marrow (Fig. 3-5).147 These stromal cells have generally been considered to be fibroblast-like, but they also display smooth muscle characteristics in culture and have been classified as myofibroblasts by some investigators.122 The particular stromal cells that support the endothelium of the venous sinuses are termed adventitial stromal cells (Fig. 3-6).147 Granulopoiesis also occurs primarily on the surface of stromal cells.125 Adi-pocytes develop from mesenchymal stem cells and may share common hematopoietic functions with stromal cells.47 Auto-nomic nerves occur in bone marrow. Their function is not clear, but direct and indirect effects of the sympathetic nervous system on hematopoietic stem cell and hematopoietic pro-genitor cell proliferation and motility have been described.61

In addition to hematopoietic cells and developing blood cells, a number of accessory cells involved in regulating hema-topoiesis reside within the extravascular space of mammalian bone marrow. These accessory cells include macrophages, mature lymphocytes, and natural killer (NK) cells.12,31,33,85 Erythrocyte development occurs in close association with marrow macrophages.26

In contrast to other organs such as skin and intestine, where continuous new cell production occurs throughout life, hematopoietic cells and their progeny in bone marrow are not arranged in stratified layers of progressively more differenti-ated cells. Although some segregation of cell types may be visualized by microscopic examination of stained bone marrow sections, the overall impression is that bone marrow contains an unstructured mixture of cells of different lineages and stages of maturation. Nonetheless, hematopoietic cells develop in specialized microenvironmental niches within the bone marrow.

Hematopoietic Stem Cells and Progenitor CellsBeginning in midgestation and continuing throughout post-natal life, mammalian blood cells are produced continuously from HSCs within the extravascular spaces of the bone marrow. HSCs are capable of proliferation; they exhibit long-term self-renewal and differentiation. HSCs replicate only once every 8 to 10 weeks.2 The term hematopoietic progenitor cell (HPC) refers to cells that form colonies in bone marrow culture like HSCs but do not have long-term self-renewal capacities. HSCs and HPCs are mononuclear cells that cannot be distinguished morphologically from lymphocytes. The presence of a transmembrane glycoprotein termed cluster of differentiation antigen 34 (CD34) has been used to identify HSCs and early HPCs, but some HSCs (possibly inactive ones) lack CD34.43 In addition, CD34 is also present on the surface of nonhematopoietic stem cells and vascular endo-thelial cells.72,149 CD34 is believed to play a role in cell adhesion.43

The most primitive HSC has the capacity to differentiate into HPCs of all blood cell lineages and several cell types in tissue. The frequency of HSCs in the marrow is estimated to be less than 0.01% of nucleated marrow cells in adult mice and less than 0.0001% of nucleated marrow cells in adult cats.2 HSCs produce HPCs that can give rise to one or more blood cell types. Thus, HPCs are much more numerous in marrow

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FIGURE 3-3 Schematic view of a cross section of bone marrow near the central longitudinal vein. Hematopoietic cells lie in the hematopoietic compartment between the vascular sinuses that drain into the central vein. The sinus wall consists of endothelial cells (end), a basement membrane, and, in some areas, adventitial stromal cells (adv). Megakaryocytes (meg) lie against the outside of the vascular sinus wall and discharge proplatelets directly into the vascular lumen through apertures in the sinus wall. Erythroid cells are shown developing in an erythroid islet (eryth islet) around a central macrophage. Emperipolesis (emp), the entry of megakaryocyte cytoplasm by other cells, is occasionally observed.

From Weiss L. The Blood Cells and Hematopoietic Tissues. New York: Elsevier; 1984.

than are HSCs. Less than 2% of nucleated bone marrow cells in adult dogs are CD34+, but up to 18% CD34+ cells have been reported in neonatal pups.38,128

The HSC produces a common lymphoid progenitor (CLP) and a common myeloid progenitor (CMP), as shown in Figure 3-7. The CLP is believed to give rise to B lymphocytes, T lymphocytes, and NK cells.16 The CMP is believed to give rise to all nonlymphoid blood cells (see Fig. 3-7) as well as macrophages, dendritic cells, osteoclasts, and mast cells.66,89 HPCs proliferate with higher frequency than do HSCs, but the self-renewal capabilities of HPCs decrease as progressive differentiation and cell lineage restrictions occur. When mea-sured in an in vitro cell culture assay, HPCs are referred to as colony-forming units (CFUs). HPCs that rapidly proliferate, retain their ability to migrate, and form multiple subcolonies around a larger central colony in culture are called burst-forming units (BFUs).

The CMP (also called a colony-forming unit-granulocyte-erythrocyte-monocyte-megakaryocyte [CFU-GEMM]) gives rise to the megakaryocyte-erythrocyte progenitor (MkEP) and the granulocyte-monocyte progenitor (GMP). The MkEP produces megakaryocyte progenitors (MkP) and erythrocyte progenitors (EP). The GMP produces the granu-locyte progenitor (GP), the monocyte-dendritic cell progeni-tor (MDP), the basophil-mast cell progenitor (BMaP), and the eosinophil progenitor (EoP) in mice (see Fig. 3-7). However, in humans, the EoP may develop from the CMP, rather than the GMP.89

Mesenchymal Stem CellsMesenchymal stem cells (MSCs) are estimated to occur in bone marrow at a frequency of 0.001% to 0.0002% of nucle-ated marrow cells.84 Evidence suggests that the MSC lineage differentiation pathways are less strictly delineated (exhibit


FIGURE 3-4 A scanning electron micrograph of the cut surface of bone marrow showing a system of vascular sinuses originating at the periphery of the marrow (right side of field) and draining into a large vein (upper left corner). The large vein has several apertures in its wall, representing tributary venous sinuses. Hematopoietic tissue lies between the vascular sinuses.

From Weiss L. The hematopoietic microenvironment of the bone marrow: an ultrastructural study of the stroma of rats. Anat Rec. 1976;186:161-184.

FIGURE 3-5 A scanning electron micrograph from the extravascular space in rat bone marrow. Spherical hematopoietic cells are shown developing in close association with marrow stromal cells and their cytoplasmic processes.

Courtesy of Ahmed Deldar.

FIGURE 3-6 Structure of the bone marrow sinus wall. Sinus lumen (s), endothelial cell (e), basement membrane (bm), hematopoietic compartment (h), adventitial stromal cell with processes (ac).

From Alsaker RD. The formation, emergence, and maturation of the reticulo-cyte: a review. Vet Clin Pathol. 1977;6(3):7-12.

FIGURE 3-7 Simplified working model of hematopoiesis. HSC, hematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common myeloid pro-genitor; T/NKP, T lymphocyte-natural killer cell progenitor; MkEP, megakaryocyte-erythroid progenitor; GMP, granulocyte-monocyte pro-genitor; NKP, natural killer cell progenitor; TLP, T lymphocyte progeni-tor; BLP, B lymphocyte progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor; BMaP, basophil-mast cell progenitor; EoP, eosino-phil progenitor, GP, granulocyte progenitor; MDP, monocyte-dendritic cell progenitor; NK, natural killer; MaP, mast cell progenitor; CDP, common dendritic progenitor.

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injection of growth factors is one approach used to collect increased numbers of stem cells from blood for human bone marrow transplantation.46

Hematopoietic MicroenvironmentBlood cell production occurs throughout life in the bone marrow of adult animals because of the unique microenviron-ment present there. The hematopoietic microenvironment is a complex meshwork composed of stromal cells, endothelial cells, adipocytes, macrophages, subsets of lymphocytes, NK cells, osteoblasts, ECM components, and glycoprotein growth factors that profoundly affect HSC and HPC engraftment, survival, proliferation, and differentiation.1

Stromal cells and endothelial cells produce components of the ECM, including collagen fibers, basement membranes of vessels and vascular sinuses, proteoglycans, and glycoproteins. In addition to providing structural support, the ECM is important in the binding of hematopoietic cells and soluble growth factors to stromal cells and other cells in the micro-environment so that optimal proliferation and differentiation can occur by virtue of these cell-cell interactions (Fig. 3-8).1,106

Collagen fibers produced by stromal cells may not have direct stimulatory effects on hematopoiesis but rather are permissive, promoting hematopoiesis by forming a scaffolding around which the other elements of the microenvironment are organized. Hematopoietic cells can adhere to collagen types I and VI.22

Adhesion molecules (most importantly β1-integrins) on the surface of hematopoietic cells bind to ECM glycoproteins such as VCAM-1, hemonectin, fibronectin, laminin, vitronec-tin, and thrombospondin. The spectrum of the expression of adhesion molecules on hematopoietic cells that differentially bind to ECM glycoproteins varies with the type, maturity, and activation state of the hematopoietic cells. In addition to anchoring cells to a given microenvironmental niche, the binding of adhesion molecules on hematopoietic cells also

greater plasticity) than the HSC pathways.115 MSCs have the ability to differentiate into multiple lineages, including marrow stromal cells, adipocytes, osteoblasts, chondrocytes, fibroblasts, and myoblasts.95 Progenitor cells for a variety of peripheral tissue cell types are also present in bone marrow. Some studies suggest that MSCs may also produce epithelial cells, hepato-cytes, and neuronal cells.81,115 Endothelial progenitor cells are present in bone marrow and blood; however, their origin remains to be clarified. Evidence has been presented suggest-ing an association with both MSCs and HSCs.24,118

Homing of Hematopoietic Stem Cells and Progenitor Cells to the MarrowHoming is the process by which circulating HSCs and HPCs bind to the luminal surface of bone marrow endothelial cells, migrate through the endothelial cells, bind selectively to sites in the extravascular space, and begin the process of prolifera-tion and differentiation. Homing of HSCs/HPCs is mediated by chemoattractants produced by endothelial cells and other cells in the microenvironment and by adhesion molecules expressed on the surfaces of HSCs/HPCs that bind to pro-teoglycans and glycoproteins on the surfaces of various marrow cells and the extracellular matrix.27

The chemokine (chemoattractant cytokine) CXCL12, also called stromal cell-derived factor-1 (SDF-1), is especially important in the homing of HSCs/HPCs, but other chemoat-tractants are also involved in this process. SDF-1 is produced by both marrow endothelial cells and stromal cells, and migra-tion of HSCs/HPCs from blood to bone marrow occurs toward an SDF-1 gradient by virtue of an SDF-1 receptor CXCR4 expressed on these migrating cells. SDF-1 promotes the expression of CXCR4 and other adhesion molecules on the surface of HSCs/HPCs and induces transendothelial migration.27

HSCs/HPCs must be activated by locally produced factors (including SDF-1) for optimal transendothelial migration to occur. P- and E-selectin molecules (membrane-spanning, sugar-binding glycoproteins), expressed on bone marrow endothelial cells, bind to glycosylated ligands on HSCs/HPCs to promote an initial loose, rolling-type adhesion between HSCs/HPCs and endothelial cells in blood. Tight adhesion and migration through endothelial cells is dependent on integrin molecules—particularly the α4β1-integrin (very late antigen-4, VLA-4) on the surfaces of migrating cells—binding to their counterreceptors, especially vascular cell adhesion molecule-1 (VCAM-1), on endothelial cells.27

The first successful bone marrow transplants were done experimentally in dogs in the late 1950s.6 Because of the homing properties of HSCs, bone marrow transplants are performed by injecting bone marrow cells into the blood. In addition, HSCs/HPCs naturally circulate in blood. The physi-ologic mechanisms involved in the release of these hemato-poietic cells from the bone marrow are not well defined, but HSC and HPC numbers can be increased markedly in blood following injection of growth factors such as granulocyte colony-stimulating factor (G-CSF).113 In fact, intravenous

FIGURE 3-8 Interactions between a progenitor cell and a stromal cell in the extravas-cular microenvironment of the bone marrow. VCAM-1, vascular cell adhesion molecule-1.


cells, and T lymphocytes to produce HGFs. Different combi-nations of HGFs regulate the growth of different types of HSCs and/or HPCs.66

Early-acting HGFs are involved with triggering dormant (GO) primitive HSCs to begin cycling. Stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt3L), and TPO are important early factors that act in combination with one or more other cytokines such as IL-3, IL-6, IL-11, and G-CSF.

Intermediate-acting HGFs have broad specificity. IL-3 (multi-CSF), granulocyte-macrophage-CSF (GM-CSF), and IL-4 support proliferation of multipotent HPCs. These factors also interact with late-acting factors to stimulate the prolifera-tion of a wide variety of committed progenitor cells. Late-acting HGFs have restricted specificity. Macrophage-CSF (M-CSF), G-CSF, EPO, TPO, and IL-5 are more restrictive in their actions. They have their most potent effects on com-mitted progenitor cells and on later stages of development when cell lines can be recognized morphologically.67 TPO appears to be an exception. In addition to stimulating platelet production, it is important in maintaining a population of HSCs in their osteoblastic niche.8

ERYTHROPOIESISPrimitive ErythropoiesisPrimitive erythropoiesis begins and predominates in the yolk sac but also occurs later in the liver. Primitive erythrocytes are large (more than 400 fL in humans), generally nucleated cells with high nuclear:cytoplasmic ratios. Their nuclei have open (noncondensed) chromatin and their cytoplasm contains pre-dominantly embryonal hemoglobin (Hb) with a high oxygen affinity.117,133,138 In mammals as in nonmammalian species, primitive RBCs enter the blood as nucleated cells, but in contrast to nonmammalian species, enucleation can eventually occur in the circulation.70 These extruded nuclei circulate for a short time in the blood. They are surrounded by a small amount of cytoplasm and have been called pyrenocytes.97

A switch to definitive erythropoiesis occurs during fetal development. This results in the production of smaller cells that generally extrude their nuclei before entering the blood, produce fetal Hb (in some species) and adult Hb, and are highly dependent on EPO for proliferation.138

Hematopoietic Progenitor Cells and the Bone Marrow MicroenvironmentThe CMP gives rise to the MkEP, which can differentiate into megakaryocyte progenitors (MkPs) or erythroid progenitors (EPs). The production of EPs is stimulated by SCF, IL-3, GM-CSF, and TPO.67,78 The earliest EP is the burst-forming-unit erythrocyte (BFU-E), which differentiates into the colony-forming-unit erythrocyte (CFU-E). EPO is the primary growth factor involved in the proliferation and dif-ferentiation of CFU-Es into rubriblasts, the first morphologi-cally recognizable erythroid cells. CFU-Es are more responsive to EPO than BFU-E cells because CFU-Es exhibit greater numbers of surface receptors for EPO.116

plays a role in cell regulation directly by activating signal pathways for cell growth, survival, and differentiation or indi-rectly by modulating the responses to hematopoietic growth factors.22

A proteoglycan consists of a protein core with repeating carbohydrate glycosaminoglycans (GAGs) attached. Major proteoglycans in the marrow include heparan sulfate, chon-droitin sulfate, hyaluronic acid, and dermatan sulfate. Proteo-glycans enhance hematopoiesis by trapping soluble growth factors in the vicinity of hematopoietic cells and by strength-ening the binding of hematopoietic cells to the stroma.28

Hematopoietic cells develop in specific niches within the marrow. During steady-state conditions, quiescent HSCs are concentrated near endosteal and trabecular bone, where osteo-blasts help to regulate their numbers.152 HSCs and HPCs are also located near vascular sinuses, where they appear more active. HSCs and HPCs in this vascular niche likely have homeostatic roles during steady-state conditions.94 Erythroid cells develop around macrophages and megakaryocytes form adjacent to sinusoidal endothelial cells; granulocyte develop-ment is associated with stromal cells located away from the vascular sinuses.1,63,66

Hematopoietic Growth FactorsProliferation of HSCs and HPCs cannot occur spontaneously but requires the presence of specific hematopoietic growth factors (HGFs); these may be produced locally in the bone marrow (paracrine or autocrine) or more remotely by periph-eral tissues and transported to the marrow through the blood (endocrine). All cells in the hematopoietic microenvironment, including the hematopoietic cells themselves, produce HGFs and/or inhibitors of hematopoiesis.69 Some HGFs have been called poietins (erythropoietin [EPO] and thrombopoietin [TPO]). Other growth factors have been classified as colony-stimulating factors (CSFs) based on in vitro culture studies. Finally, some HGFs have been described as interleukins (ILs).67

Hematopoietic cells express receptors for more than one HGF on their surfaces. The number of each receptor type present depends on the stage of cell activation and differentia-tion. Binding of an HGF to its receptor results in a series of enzymatic reactions that generate transcription factors; these promote the synthesis of molecules that inhibit apoptosis, the formation of cell-cycle regulators (cyclins), and the synthesis of additional HGFs and their receptors.22,67 The pathways involved in generating lineage-restricted transcription factors is complex and beyond the scope of this text.22

HGFs vary in the type(s) of HSCs and/or HPCs that they can stimulate to proliferate. Factors are often synergistic in their effects on hematopoietic cells. In some instances, an HGF may not directly stimulate the proliferation of a given cell type, but may potentiate its proliferation by inducing the expression of membrane receptors for HGFs that do directly stimulate proliferation. Some glycoproteins, such as IL-1 and tumor necrosis factor-α (TNF-α), can modulate hematopoi-esis indirectly by stimulating marrow stromal cells, endothelial


progressively accumulates, imparting a red coloration to the cytoplasm (Fig. 3-10). Cells with both red and blue coloration are described as having polychromatophilic cytoplasm. An immature erythrocyte, termed a reticulocyte, is formed follow-ing extrusion of the metarubricyte nucleus. This generally occurs while cells are still bound to central macrophages.26 Extruded nuclei are bound and phagocytosed by a novel receptor on the surface of bone marrow macrophages.107 However, nuclei can be extruded in blood when metarubri-cytes are released from the bone marrow (Fig. 3-11).119

Early reticulocytes have polylobulated surfaces. Their cyto-plasm contains ribosomes, polyribosomes, and mitochondria necessary for the completion of Hb synthesis.15 Reticulocytes derive their name from a network or reticulum that appears when they are stained with basic dyes such as new methylene blue and brilliant cresyl green. That network is not preexisting but rather an artifact formed by the precipitation of ribosomal ribonucleic acids and proteins secondary to staining.57 As reticulocytes mature, the amount of ribosomal material decreases until only a few basophilic specks can be visualized with reticulocyte staining procedures. These mature reticulo-cytes have been referred to as type IV reticulocytes53 or punc-tate reticulocytes.7,101

The development of a reticulocyte into a mature erythro-cyte is a gradual process that requires a variable number of days depending on the species involved. Consequently the morphologic and physiologic properties of reticulocytes vary with the stage of maturation. The cell surface undergoes extensive remodeling, with loss of membrane material and ultimately the formation of the biconcave shape of mature

Marrow macrophages are important components of the hematopoietic microenvironment involved with erythropoie-sis. Both early and late stages of erythroid development occur with intimate membrane apposition to central macrophages in “erythroid islands.” Several adhesion molecules on ery-throid cells and macrophages, and extracellular matrix glyco-proteins are important in forming these erythroid islands.26 Direct contact with these macrophages enhances the prolif-eration of erythroid precursors under basal conditions. Central macrophages may promote basal erythrocyte production by producing positive growth factors, including EPO; however, they may inhibit erythropoiesis by producing negative factors such as IL-1, TNF-α, transforming growth factor-β (TGF-β), and interferons (IFNs)-α, -β, and -γ in inflammatory conditions.25,145,154 The finding that EPO can also be produced by erythroid progenitors suggests that these cells may support erythropoiesis by autocrine stimulation.126 Although some degree of basal regulation of erythropoiesis occurs within the marrow microenvironment, humoral regulation is also impor-tant, with EPO production occurring primarily within peri-tubular interstitial cells of the kidney and various inhibitory cytokines being produced at sites of inflammation throughout the body.

Nutrients Needed for ErythropoiesisIn addition to amino acids and essential fatty acids, several metals and vitamins are required for normal erythropoiesis. Iron is needed for the synthesis of heme, an essential compo-nent of Hb and certain enzymes. Copper, in the form of ceruloplasmin, is important in the release of iron from tissue to plasma for transport to developing erythroid cells. Vitamin B6 (pyridoxine) is needed as a cofactor in the first enzymatic step in heme synthesis.

Tetrahydrofolic acid, the active form of folic acid (a B vitamin), is needed for the transfer of single carbon-containing molecules in DNA and RNA synthesis. The physiologic mechanism of B12 involvement in erythrocyte production is not well understood, but it is related to folate metabolism. Cobalt is essential for the synthesis of B12 by ruminants.51

Maturation of Erythroid CellsRubriblasts are continuously generated from progenitor cells in the extravascular space of the bone marrow. The production of a rubriblast initiates a series of approximately four divisions over a period of 3 or 4 days to produce about 16 metarubri-cytes that are no longer capable of division (Fig. 3-9).36 These divisions are called maturational divisions because there is a progressive maturation of the nucleus and cytoplasm con-comitant with each division.

When they are stained with Romanowsky-type blood stains, early precursors have intensely blue cytoplasm owing to the presence of many basophilic ribosomes and polyribo-somes that are actively synthesizing globin chains and smaller amounts of other proteins. As these cells divide and mature, overall cell size decreases, nuclear chromatin condensation increases, cytoplasmic basophilia decreases, and Hb

FIGURE 3-9 Diagram of erythropoiesis showing the release of reticulocytes into blood as it normally occurs in dogs.

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FIGURE 3-10 Maturation of canine erythroid and granulocytic cells as they appear in Wright-Giemsa-stained bone marrow aspirate smears.

Drawing by Perry Bain.

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erythrocytes.15 The loss of membrane protein and lipid com-ponents requires ATP and involves the formation of intracel-lular multivesicular endosomes that fuse with the plasma membrane, releasing vesicles (exosomes) extracellularly.45,140 This is a highly selective process in which some proteins (e.g., transferrin receptor 1 and fibronectin receptor) are lost and cytoskeletal proteins (e.g., spectrin) and firmly bound trans-membrane proteins (e.g., the anion transporter and glycopho-rin A) are retained and concentrated.45,103

The mitochondria undergo degenerative changes in a pro-grammed death phenomenon (mitoptosis)45 and are either digested or extruded following entrapment in structures resembling autophagic vacuoles (Fig. 3-12).90,120 The poly-somes separate into monosomes, decrease in number, and disappear as reticulocytes mature into erythrocytes. The deg-radation of ribosomes appears to be energy-dependent and presumably involves proteases and RNAases.112

Reticulocyte maturation begins in the bone marrow and is completed in the peripheral blood and spleen in dogs, cats, and pigs.56 As reticulocytes mature, they lose the surface receptors needed to adhere to the fibronectin and thrombo-spondin components of the extracellular matrix, presumably facilitating their release from the bone marrow.132

Reticulocytes become progressively more deformable as they mature, a characteristic that also facilitates their release from the marrow.144 To exit the extravascular space of the marrow, reticulocytes press against the abluminal surfaces of endothe-lial cells making up the sinus wall. Cytoplasm thins and small pores develop in endothelial cells, which allow reticulocytes to be pushed through by a small pressure gradient across the sinus wall.79,143 These pores apparently close after cell passage.

Relatively immature aggregate-type reticulocytes are released from canine bone marrow; consequently most of these cells appear polychromatophilic when they are viewed following routine blood-film staining procedures.73 Reticulo-cytes are generally not released from bone marrow of non-anemic cats until they mature to punctate-type reticulocytes (Fig. 3-13); consequently few or no aggregate reticulocytes (less than 0.4%) but up to 10% punctate reticulocytes are found in blood from normal adult cats.29 The high percentage of punctate reticulocytes results from a long maturation time with delayed degradation of RNA.39 Reticulocytes are gener-ally absent in the peripheral blood of healthy adult cattle and goats, but a small number of punctate types (0.5%) may occur in adult sheep.56 Based on microscopic examination of blood films stained with new methylene blue, equine reticulocytes


FIGURE 3-11 Nuclear extrusion of metarubricytes to form canine reticulocytes. A, Blood film from a dog with a hemolytic anemia secondary to hemangio-sarcoma. Frictional forces during smear preparation may have contrib-uted to the nuclear extrusion. B, Transmission electron microscopy of nuclear extrusion of a metarubricyte.

B, From Simpson CF, King JM. The mechanism of denucleation in circulating erythroblasts. J Cell Biol. 1967;35:237-245.

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FIGURE 3-12 Transmission electron microscopy of mitochondrial extrusion from canine reticulocytes. A, A series of vesicles appear to be surrounding three mitochondria. B, Fusion of a vacuole containing mitochondria with the reticulocyte outer membrane, thereby promoting mitochondrial extrusion.

From Simpson CF, King JM. The mechanism of mitochondrial extrusion from phenylhydrazine-induced reticulocytes in the circulating blood. J Cell Biol. 1968;36:103-109.

FIGURE 3-13 Cat erythroid cells demonstrating reticulocyte release into blood as it occurs in most normal cats. Note that punctate reticulocytes do not appear polychromatophilic when stained with Wright-Giemsa.

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are normally absent from blood and are rarely released in response to anemia.

Control of ErythropoiesisEarly- and intermediate-acting growth factors—including SCF, IL-3, GM-CSF, and TPO—are utilized to produce EPs. EPO is the principal growth factor promoting the viability, proliferation, and differentiation of EPs (BFU-E and CFU-E) that express specific cell-surface EPO receptors. The main mechanism used to achieve these effects is inhibition of apop-tosis.124 Early BFU-E cells do not express EPO receptors, but more mature BFU-E cells do and are thus responsive to EPO. EPO receptor numbers on cell surfaces increase to maximum

values in CFU-E cells, decline in rubriblasts, and continue to decrease in the later stages of erythroid development.104,105 Because of their EPO receptor density, CFU-E cells readily respond to EPO, promoting their proliferation, differentia-tion, and transformation into rubriblasts, the first morpho-logically recognizable erythroid cell type. High concentrations of EPO may accelerate rubriblast entry into the first mitotic division, thus shortening the marrow transit time and result-ing in the early release of stress reticulocytes.105

In the presence of EPO, other hormones—including androgens, glucocorticoid hormones, growth hormone, insulin,


of vascular tone, and exerting cardioprotective and neuropro-tective effects.59

LEUKOPOIESISNeutrophil ProductionNeutrophilic cells within the bone marrow can be included in two pools (Fig. 3-15). The proliferation and maturation pool (mitotic pool) includes myeloblasts, promyelocytes, and myelocytes. Approximately four or five divisions occur over several days (Fig. 3-16). During this time primary (reddish purple) cytoplasmic granules are produced in late myeloblasts or early promyelocytes and secondary (specific) granules are synthesized within myelocytes (see Fig. 3-10). Once nuclear indentation and condensation become apparent, precursor

and insulin-like growth factors (IGFs)—can enhance the growth of erythroid progenitor cells in vitro.76,88 The thyroid hormone 3,5,3′-triiodothyronine (T3) promotes the differen-tiation and maturation of erythroid cells.76 Thyroid hormones may also promote the synthesis of EPO in the kidney.82 EPO production in adult mammals occurs primarily within peritu-bular interstitial cells located within the inner cortex and outer medulla of the kidney. The liver is an extrarenal source of EPO in adults and the major site of EPO production in the mam-malian fetus.59 Bone marrow macrophages and erythroid pro-genitor cells themselves can produce EPO, suggesting the possibility of short-range regulation of erythropoiesis.126,141

Hematopoietic cells die not only as a consequence of lack of HGFs but also in response to the presence of molecules that induce apoptosis. Inhibitors of erythropoiesis include TGF-β, TNF-α, IFN-γ, IL-6, and TNF-related apoptosis-inducing ligand (TRAIL).26

The ability to deliver oxygen to the tissues depends on cardiovascular integrity, oxygen content in arterial blood, and Hb oxygen affinity. Low oxygen content in the blood can result from a low partial pressure of oxygen (PO2) in arterial blood, as occurs at high altitudes or with congenital heart defects in which some of the blood flow bypasses the pulmo-nary circulation. A low oxygen content in blood can also be present when arterial PO2 is normal, as occurs with anemia and methemoglobinemia. An increased oxygen affinity of Hb within erythrocytes results in a decreased tendency to release oxygen to the tissues.86 Regardless of the cause, EPO produc-tion is stimulated by tissue hypoxia (Fig. 3-14), which is mediated by hypoxia-inducible factors that control the tran-scription of the EPO gene in EPO-secreting cells.50,59

Other tissues also exhibit EPO receptors, and EPO stimu-lates nonhematopoietic actions, including promoting the proliferation and migration of endothelial cells, enhancing neovascularization, stimulating the production of modulators

FIGURE 3-14 Central role of erythropoietin (EPO) in the control of erythropoiesis. BFU-E, burst-forming unit-erythroid; CFU-E, colony-forming unit-erythroid; NRBC, nucleated red blood cell.

Marrow

Kidney

Oxygen sensor

O2 consumptionBlood flow

O2 supplyBlood flowOxygen contentOxygen affinity

EPO RBC

BFU-E CFU-E NRBC

FIGURE 3-16 A diagram of granulopoiesis.

Myeloblast

Marrowrelease

Promyelocytes

Myelocytes

Myelocytes

Myelocytes

Metamyelocytes

Bands

Neutrophils

Neutrophils

FIGURE 3-15 Approximate sizes of mitotic and postmitotic neutrophilic compartments within bone marrow.

Bone marrow

Blast Myelo Meta Band Segmented

Pro

Blood

Mitosis Maturation and storage

CNP

MNP


neutrophilic colony formation. If this also happens in vivo, it might provide negative feedback for neutrophil production in the extravascular space of bone marrow.71 One possible mech-anism is the release of serine proteases, such as elastase, from neutrophils. Elastase appears to inhibit granulopoiesis by inactivating G-CSF.35 Second, increased neutrophil numbers in blood are associated with the increased clearance of circu-lating G-CSF following binding to surface receptors on neu-trophils, thereby decreasing the primary stimulus for their production.65,75 Third, mature neutrophils indirectly inhibit granulopoiesis by the removal (through phagocytosis) of invading microorganisms that would otherwise stimulate the production of HGFs by tissue cells. Activated T lymphocytes may also inhibit neutrophil production by secreting the soluble molecule Fas-ligand and the cytokine IFN-γ.99

Eosinophil, Basophil, and Mast Cell ProductionThe eosinophil progenitor (EoP) or colony-forming unit eosinophil (CFU-Eo) is reported to develop from the CMP in humans but downstream from the GMP in mice.89 Eosino-phil production in the marrow parallels that of neutrophils. Eosinophil precursors become recognizable at the myelocyte stage, when their characteristic secondary granules appear (see Fig. 3-10). The marrow transit time is 1 week or less, with a significant storage pool of mature eosinophils.142 As in the case of neutrophils, growth factors, including IL-3 and GM-CSF, are needed for the proliferation of early progeni-tors. In addition, activated TH2 lymphocytes produce IL-5, which promotes the terminal maturation of eosinophils. IL-3, GM-CSF, and IL-5 also inhibit eosinophil apoptosis,62 while inhibitors of eosinophil production include IL-12 and IFN-γ.110

The GMP reportedly produces the bipotential basophil-mast cell progenitor (BMaP), which gives rise to the basophil progenitor and the mast cell progenitor (MaP).121 Like eosin-ophils, basophil precursors become recognizable at the myelo-cyte stage, when their characteristic secondary granules appear (see Fig. 3-10). A specific growth factor for the production of basophils has not been identified. IL-3 appears to be the major growth and differentiation factor for basophils, but other growth factors—including GM-CSF, IL-5, TGF-β, and nerve growth factor—also promote the production of basophils.37

In contrast to basophils, which mature in the bone marrow, maturation of mast cell progenitors into mast cells occurs in the tissues.40 SCF appears to be the major growth and dif-ferentiation factor for mast cells. Additional cytokines—including IL-3, IL-4, IL-9, IL-10, and IL-13—also stimulate mast cell production.54 Some local proliferation of mast cells can occur in tissues if they are appropriately stimulated.41

Production of Monocytes, Macrophages, Dendritic Cells, and OsteoclastsBone marrow MDPs give rise to monocytes and common dendritic cell progenitors (CDPs).153 Monocytes are produced through the combined effects of IL-3, GM-CSF, M-CSF, and

cells are no longer capable of division. The maturation and storage pool (postmitotic pool) includes metamyelocytes, bands, and segmented neutrophils. Cells within this pool nor-mally undergo maturation and storage for several more days prior to the migration of mature neutrophils through the vascular endothelium and into the circulation.123 The number of mature neutrophils stored in marrow is more than seven times the number present in the circulation of the dog.32 The marrow transit time from myeloblast to release of mature neutrophils into the blood varies by species but is generally between 6 and 9 days. This time can be shortened considerably when inflammation is present.108,123

A variety of cytokines with overlapping specificities are important in neutrophil production (also called granulopoie-sis). IL-3, GM-CSF, and G-CSF are of primary importance in the production of neutrophils. These cytokines act on various stages of development from CMPs to GMPs to GPs, depending on the array of growth factor receptors displayed on their surfaces. GPs are stimulated to proliferate and dif-ferentiate into myeloblasts by G-CSF. This cytokine appears to play a role in the basal regulation of granulopoiesis as well as to function as a primary regulator of the neutrophil response to inflammatory stimuli. G-CSF increases the number of cell divisions and reduces the time for granulocytic progenitors to develop into neutrophils. It also promotes the release of neu-trophils from bone marrow into blood.108,127

As neutrophils mature, there is a progressive downregula-tion of certain surface receptors, including CXCR4 and the α4β1 integrin, that adhere neutrophils to glycoproteins within the extravascular space. CXCR4 binds to CXCL12/SDF-1 produced by stromal cells, and the α4β1 integrin binds to VCAM-1 on endothelial cells. Experimental neutralization of CXCR4 and VCAM-1 results in an increased release of neu-trophils into blood. G-CSF promotes neutrophil release from bone marrow at least in part by decreasing CXCL12/SDF-1 production and decreasing CXCR4 expression on the surface of neutrophils.127

Activated helper T lymphocytes produce various growth factors including IL-3 and GM-CSF. Mononuclear phago-cytes, fibroblasts, and endothelial cells can also produce GM-CSF and G-CSF when appropriately stimulated. Mono-nuclear phagocytes can not only synthesize HGFs when they contact bacterial products but can also stimulate other cells to produce them. The cytokines, IL-1 and TNF-α, produced by monocytes and macrophages stimulate the production of HGFs by other cell types. These monokines are important in the inflammatory response to foreign organisms and neoplas-tic cells, but their role in resting granulopoiesis is unclear.66,123 IL-6 is a multifunctional cytokine that regulates inflammation (including the hepatic acute-phase response), the immune response, and hematopoiesis. In this latter role, it promotes granulopoiesis and thrombopoiesis during inflammation.111

Inhibition of neutrophil production is not well understood, but mature neutrophils may provide negative feedback inhibi-tion on their own production in three ways. First, the addition of mature neutrophils to bone marrow culture inhibits


required for pre-B lymphocytes to develop into mature, naive B lymphocytes in the marrow and enter the circulation. Less than 20% of B lymphocytes produced in the marrow become part of the peripheral mature B lymphocyte pool, with most of the cells being culled in the bone marrow or after their entry into blood.83 B lymphocytes also proliferate in peripheral lymphoid tissues in adults. As with other blood cells, the microenvironment of the marrow and lymphoid organs is important for lymphopoiesis. The production of antigen-sensitive, surface-immunoglobulin-positive B lym-phocytes is marked by successive rearrangements of the immunoglobulin gene loci and selective expression of surface proteins. Although a number of cytokines—including SCF, Flt3L, SDF-1, and IGF—are involved in B lymphocyte pro-duction in marrow, IL-7 appears to be an especially important positive growth factor.19,77 B lymphocyte lymphopoiesis is inhibited by several factors, including TGF-β, IFN-α, IFN-β, and IFN-γ.77

Recirculating B lymphocytes are activated by antigenic stimulation in the T lymphocyte region of secondary lym-phoid organs, followed by migration to the cortex in lymph nodes and to follicles in jejunal Peyer’s patches and the spleen in mammals.135 B lymphocyte activation and differentiation into plasmablasts is induced by combinations of microbial products, cytokines, and molecules bound to the surfaces of T lymphocytes and dendritic cells. Plasmablasts can develop into plasma cells in the lymphoid organs where they are pro-duced or can migrate through blood and develop into plasma cells in peripheral tissues or bone marrow. SDF-1 attracts circulating plasmablasts to the bone marrow, and factors including SDF-1 and IL-6 promote plasma cell development by preventing apoptosis.91

T lymphocyte progenitors leave the marrow and migrate to the thymus. Homing of these cells to the thymus depends on their interaction with various adhesion molecules on thymic endothelial cells and the production of specific che-motactic factors by thymic stromal cells. T lymphocyte pro-genitors develop into T lymphocytes under the influence of the thymic microenvironment and growth factors (including Flt3L and IL-7) produced in the thymus.151 After maturation in the thymus, T lymphocytes accumulate within paracortical areas of lymph nodes, periarteriolar lymphoid sheaths of the spleen, and the interfollicular areas of jejunal Peyer’s patches in mammals.135

Most NK cells are produced from progenitor cells in the bone marrow, where they undergo expansion and maturation for a week or more before their release into the blood.155 Growth factors controlling their production need further characterization, but SCF, IL-2, IL-7, and IL-15 can stimu-late NK cell development from progenitor cells in vitro.3 Subsets of NK cells also develop in the thymus and possibly other organs, such as lymph nodes, liver, and spleen. These sites may depend on the trafficking of bone marrow–derived progenitor cells and/or immature NK cells into these organs from the blood, where they mature under the influence of microenvironmental factors.49,55

IL-34 on the proliferation and differentiation of bone marrow progenitor cells.44 Less time is required to produce monocytes than granulocytes, and there is little marrow reserve of these cells.

Monocytes have long been viewed primarily as precursors that develop into tissue macrophages and dendritic cells. However, it is now recognized that many macrophage and dendritic cells in tissues do not originate from monocytes under steady-state conditions because these cells are capable of self-replication. In addition, neither microglia (macro-phages in the central nervous system) nor Langerhans cells (epidermal dendritic cells) depend on cells from the bone marrow for their renewal under steady-state conditions and possibly also during inflammation.10 In fact, Langerhans cells appear to develop from embryonic progenitor cells that enter the epidermis before birth.44

Monocytes are important effector cells during inflamma-tory conditions. They exit the blood, respond to the tissue environment, and differentiate into subsets of macrophages and inflammatory dendritic cells. Exposure to M-CSF pro-motes the development of monocytes into macrophages. The addition of IFN-γ to M-CSF promotes the formation of M1-like macrophages, while the addition of IL-4 to M-CSF induces the differentiation of M2-like macrophages. The exposure of monocytes to GM-CSF, IL-4, and TNF-α pro-motes their development into inflammatory dendritic cells or TNF-α and inducible nitric oxide synthase (iNOS)-producing (TiP)-dendritic cells.10,44,153

CDPs can give rise to preclassic dendritic cells (Pre-cDC) and plasmacytoid dendritic cells (pDCs) in bone marrow.96 Both cell types are released into blood and enter the tissues, where the Pre-cDCs develop into classic dendritic cells (cDC) in lymphoid organs and mucosal dendritic cells.44,153 Cytokines—including Flt3L, GM-CSF, and lymphotoxin α1β2—appear to be important for the development of cDCs and pDCs.10,44

Osteoclasts develop when monocyte progenitors are cul-tured with M-CSF and a soluble form of receptor activator of nuclear factor-κB ligand (RANKL).9 IL-3 and GM-CSF inhibit osteoclast formation. The relative amounts of these growth factors and presumably others present in the micro-environment of a monocyte progenitor apparently determine whether macrophages, dendritic cells, or osteoclasts are formed.

Lymphocyte and NK Cell ProductionThe CLP is believed to give rise to B lymphocytes, T lym-phocytes, and NK cells.16 The development of B lymphocyte and T lymphocyte progenitors in bone marrow is antigen-independent. Both SCF and Flt3L appear to be involved in the production of early lymphoid progenitor cells in mice.17

B lymphocyte progenitors produce mature, naive B lym-phocytes in the marrow in most mammals, in specialized ileal Peyer’s patches in dogs, pigs, and ruminants, and in the bursa of Fabricius in birds.83,135 Approximately 2 to 3 days are


individual platelets within the sinuses and general circula-tion.60 Megakaryocytes may rarely migrate through the vas-cular endothelium into the sinuses, enter the general venous circulation (see Fig. 2-34), and become lodged in pulmonary capillaries.11 It is estimated that 1000 to 3000 platelets are produced from each megakaryocyte, depending on mega-karyocyte size.68 Megakaryocytes are not present in nonmam-malian species. Like erythrocytes and leukocytes, the nucleated thrombocytes of nonmammalian species are produced by mitosis of precursor cells.

A number of cytokines can stimulate or enhance the pro-liferation and expansion of megakaryocyte progenitor cells. Factors that may be involved include SCF, Flt3L, IL-3, GM-CSF, IL-11, and EPO.13,18,68 TPO is the key stimulator of platelet production by stimulating megakaryocyte prolifera-tion, survival, and size (ploidy).60,68 TPO also transiently enhances the aggregatory response of platelets to agonists.4

Although various cells in the body can produce TPO, including cells in the kidney and bone marrow stromal cells,80,87 the major sites of TPO production appear to be the endothelial cells of the liver.58,148 The amount of TPO pro-duced in the body appears to be relatively constant. TPO receptors (c-Mpl receptors) on blood platelets and maturing megakaryocytes can bind, internalize, and degrade TPO, providing negative feedback on platelet production.68 Conse-quently blood TPO concentration is remarkably high in the case of thrombocytopenia resulting from megakaryocytic hypoplasia. In contrast, blood TPO concentrations are much lower with ongoing immune-mediated thrombocytopenia, because megakaryocytes are generally increased in the marrow and rapid platelet turnover is occurring, resulting in increased binding and removal of TPO from blood.52

However, the number of maturing megakaryocytes and blood platelets present may not be the only determinants of blood TPO concentrations. IL-6 stimulates thrombopoiesis by increasing the production of TPO by the liver, which con-tributes to the thrombocytosis seen in some inflammatory conditions.64 Conversely, platelet factor 4 (PF4), TGF-β, IL-4, and TNF-α appear to be inhibitors of megakaryocyte production.13,137

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